Abstract
Two isolated recombinant fragments from human integrin alpha(5)beta(1) encompassing the FG-GAP repeats III to VII of alpha(5) and the insertion-type domain from beta(1), respectively, are structurally well defined in solution, based on CD evidence. Divalent cation binding induces a conformational adaptation that is achieved by Ca(2+) or Mg(2+) (or Mn(2+)) with alpha(5) and only by Mg(2+) (or Mn(2+)) with beta(1). Mn(2+) bound to beta(1) is highly hydrated ( approximately 3 water molecules), based on water NMR relaxation, in agreement with a metal ion-dependent adhesion site-type metal coordination. Each fragment saturated with Mg(2+) (or Mn(2+)) binds a recombinant fibronectin ligand in an RGD-dependent manner. A conformational rearrangement is induced on the fibronectin ligand upon binding to the alpha(5), but not to the beta(1) fragment, based on CD. Ligand binding results in metal ion displacement from beta(1). Both alpha(5) and beta(1) fragments form a stable heterodimer (alpha(5)beta(1) mini-integrin) that retains ligand recognition to form a 1:1:1 ternary complex, in the presence of Mg(2+), and induces a specific conformational adaptation of the fibronectin ligand. A two-site model for RGD binding to both alpha and beta integrin components is inferred from our data using low molecular weight RGD mimetics.
Highlights
Integrins (IN)1 are a family of structurally and functionally related adhesion receptors that participate in cell-cell and cellextracellular matrix (EM) interactions (1)
This I-type domain in the integrin  subunits includes the following: (i) a functional cation-binding site that displays strong similarities at the level of its metal-coordinating residues with the MIDAS site of the ␣ subunit I-domains (13, 14); (ii) a totally conserved DDL motif, close to the MIDAS-type site that is apparently involved in the recognition of RGD-containing ligands (15); (iii) possibly a specific sequence responsible for heterodimer formation; in the case of the human 3 I-type domain this specific sequence corresponds to the segment 3(275–280) with the unique hexapeptide sequence, VGSDNH, that is responsible for species-restricted ␣ heterodimer assembly (16)
A model has been recently proposed (10) in which the RGD tripeptide sequence from the protein ligand acts as a dual motif with two oppositely charged groups, a negative one and a positive one, that interact with distinct structural elements on each of both ␣ and  subunits
Summary
Materials and Buffers—Buffer A contained 12.5 mM sodium borate, 50 mM KCl at pH 8.3. Buffer B contained 20 mM potassium cacodylate, 50 mM KCl at pH 6.0. The PCR fragment was purified using the Qiaquick gel extraction kit (Qiagen, Chatsworth, CA), digested with NcoI and BamHI, and inserted into the pET32a vector (Novagen, Madison, WI) This vector allows expression of the corresponding sequence fused with thioredoxin (Trx) and a hexahistidinyl segment at the N terminus of the target protein. After centrifugation (80,000 ϫ g for 30 min), the recombinant IN and Fn molecules were mixed at a 1:1 molar ratio (at protein concentrations in the 10 M range), incubated at room temperature for 2 h, and subjected to chemical cross-linking by addition of dithiobis(succinimidyl propionate) (Pierce; 125 mM stock solution in N,N-dimethylformamide) to a final concentration of 0.5 mM (40). The protein concentration was kept nearly constant over the entire range of cation concentrations (the maximum dilution effect did not exceed 2% of the initial protein concentration)
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